Abstract

The
anti-inflammatory activities of extract of Calotropis giganteaR.Br.and Tridax procumbens
Linn., were assessed on carrageenin-induced paw edema along with standard
drug, Ibuprofen. The Ibuprofen significantly reduced paw
edema at the dose of 200mg/Kg bw orally. The oral administration
equi-effective dose (ED50) of C. gigantea (600mg/Kg bw) and
T. procumbens (400 mg/Kg bw) individually revealed about 20-35% more activity
than the one rendered by administration of 50mg/Kg bw of Ibuprofen. The effect of C.
gigantea and T. procumbens along with various dose regimen of Ibuprofen showed greater
anti-inflammatory activities than the Ibuprofen
alone.

����� Inflammation is a common reaction of
the body to be insult cause by various biological and non-biological factors
present in the environment. In our country there are a large number of people
suffering of deprivation of even essential need, and the �urge to survive�
has prompt them to explore naturally available resources for therapeutic
effects with respect to common ailments including inflammation. This has
allowed to development of alternative traditional method of therapy. Calotropis
gigantea R.Br.
(Asclepiadaceae)and T.
procumbens Linn. (Compositae),known as Arka and Jayanti in ayurveda, have been widely documented
in the ayurvedic and traditional medical literature for various therapeutics
applications. C. gigantea has been
used as a violent purgative, gastrointestinal irritant, inducing abortion and
treatment of earache, toothache, headache, anxiety, sprain and stiff joints
to cure pain1-9. It has been also reported as analgesic activity,
anti-inflammatory and pregnancy interceptive activity2, 10-12.
Similarly, T. procumbens has also
been reported to be useful in various diseases like inflammation, hepatic
disorder, wounds, microbial infections, reduce immunity and free radical
generated diseases like arthrosclerasis, various neurological disorder13-22.
Both of these plants are widely distributed in the planes of Asia and Africa
and grow profusely in the wild1,2.

�����
Ibuprofen is an NSAID, which is believed to work through inhibition of
cyclooxygenase (COX-1 and COX-2). It�s analgesic, antipyretic and
anti-inflammatory activities are achieved principally through COX-2 inhibition23. The toxic effects
of an Ibuprofen are unlikely at doses below 100 mg/kg but can be severe above
400 mg/kg24, however, large doses do not indicate that
the clinical course is likely to be lethal25.

Material and methods:

Materials:

Calotropis giganteaR.Br.and Tridax procumbens
Linn. were collected from botanical garden of Bundelkhand University, Jhansi,
India, in month of December. The materials were taxonomically identified and
authenticated by National Botanical Research Institute, Lucknow, India and
the vouchers of specimen stored. Carrageenin and Gum acacia were purchased
from Sigma Biochemicals, Ibuprofen
from Cipla and Ethanol purchases from E-Merk.

Extraction:

T. procumbens was extracted by simple maceration process. 300g of
fresh leaves were ground with the help of mortar and pestle. Juice obtained
was filtered through muslin cloth. The filtrate was evaporated by rotary
evaporator (cryochiller). Similarly, 300g ofshed-dried leaves of C. gigantea were extracted 500ml
of 60% ethyl alcohol for 12 hour in using Soxhlet apparatus. The extraction
was filtrated and dried by rotary evaporator.

Animals:

The study used male and
female albino rats weighing 130-170g. They were kept in polypropylene cages
in centrally air conditioned room at an ambient temperature of 25�1 0C
and 12 h light and dark cycle. All animals were fed standard animal feed and
tap water ad libitum and left to
acclimatize at least for one week before beginning the experiment. All
experiments were carried out in accordance with the guideline of the CPCSEA.

Induction of inflammation:

The test was used to
determine the anti-inflammatory action of the extract by the method of Winter
et al.,26.
Paw edema was induced by an intradermal injection of carrageenin (5% in gum
acacia). Carrageenin induced rat hind paw edema has been widely used for the
discovery and evaluation of anti-inflammatory drugs; since the relative
potency estimate obtained from most drugs tend to reflect clinical experience26.
This suitable test also has frequently been used to access the anti-edematous
effect of natural products27. The local injection of carrageenan
induced inflammatory process in the rat involves three phases by several
mediators released in ordinate sequence28. An initial phase,
during the first 1.5 h, is caused by the release of histamine and seratonin,
second phase is mediated by bradykinine between 1.5 to 2.5 h and finally,
third phase, the mediator of which is possibly to prostaglandin occurring
between 2.5 to 6 h after the carrageenin injection. The third phase appears
to be the most interesting phase compared with the two earlier phases due to
the maximum vascular response as determined by leucocytes migration to
inflamed area29. It is well established that prostaglandin, by
virtue of their activity, as modulators of inflammatory responses, have a
major role in inflammatory mechanism.

Anti-inflammatory
activity:

The edema volume was
determined using a Plethysmometer prior to and first,
third and fifth hours after carrageenin injection. The drug Ibuprofen and extract were diluted
in saline and in distilled water respectively. The animal were distributed in
different groups, each group were five animals. The test drugs were given
orally one hour prior to carrageenin injection and control group received
vehicle only. In first phase, only 25, 50, 100, 150 and 200mg/Kg bw of Ibuprofen were administrated to
different group. In second phase 600mg/Kg (ED50) of C. gigantea
together with different dose regimen (as given above) of Ibuprofen administrated. Similarly,
in third phase 400mg/Kg (ED50) bw of T. procumbens along
with the above dose regimen of Ibuprofen were used.�

Results and
Discussion:

������ The anti-inflammatory activity of
orally administered Ibuprofen, Ibuprofen along with C. gigantea and T.
Procumbens on carrageenin-induced rat hind paw edema is shown in figure
1a, b, c and d. Results were expressed as mean � standard error of mean (SEM). One-way ANOVA test was applied to evaluate
the significant p values and 0.05 was considered as
significant. The 50% effective dose (ED50), calculated as
the dose required to 50% inhibition of the carrageenin-induced edema
formation and it was 600mg/Kg bw of C. gigantea and 400mg/Kg bw of
T. procumbens.

����� The standard drug Ibuprofen presented a
dose-dependent anti-inflammatory activity after carrageenin injection. The
Ibuprofen treatment dose of 200mg/Kg bw reduced the edema to 87.5% in first,
64.71% in third and 50% in fifth hours respectively. While, 150 mg/Kg bw dose
reduced it to 68.75% in first, 58.82% in third and 43.75% in fifth hours
respectively (fig1a). Hence, the Ibuprofen seems to be sensitive to
cyclooxygenase inhibitors. Cyclooxygenase has been employed to evaluate the
effect of NSAID, which primarily inhibits the cyclooxygenase involved in
prostaglandin synthesis. It has also been demonstrated that the suppression
of carrageenin-induced hind paw edema after third hour correlates reasonably
with therapeutic dose of clinically effective anti-inflammatory agents28.

������� The extract of C. gigantea at
600mg/ Kg bw inhibits the edema upto 50% in first, 41.80% in third and 25% in
fifth hours. While, T. procumbens at dose of 400mg/Kg bw inhibits upto
52.50% in first, 44.12% in third and 25% in fifth hours. The fact that the
extract inhibited edema during all phases of inflammation suggested that it
probably inhibited different aspects and chemical mediators of inflammation.
The paw edema induced by carrageenin has been extensively studied in the
assessment of the anti-inflammatory action of steroidal and non-steroidal
drugs involving several chemical mediators such as histamine, serotonin,
bradykinin and prostaglandins30. The administration of 600mg/Kg bw
of C. gigantea along with different
doses of Ibuprofen

significantly reduced paw
edema with respect to standard drug (Fig 1a,b, c,d). The dose of 100mg/Kg bw
of Ibuprofen inhibited 62.50%, 52.94% and 37.50%, while, in combination with C.gigantea inhibited 75%, 70.59% and 62.5% in first, third and fifth
hour respectively. The dose of 50mg/Kg bw of Ibuprofen inhibited 31.25%,
29.41% and 18.75% and in combination with C. gigantea the inhibition
increased from 56.25%, 47.06% and 43.75% in first, third and fifth hour
respectively. Similarly, 400mg/Kg of T. procumbens in combination with
dose of 100mg/Kg bw dose of Ibuprofen reduced the edema 75.0%, 70.59% and
62.5%, while with 50mg/Kg bw dose of Ibuprofen reduced 62.50%, 58.82% and
50.0% in first, third and fifth hour respectively.

����� The comparative analysis shown that, T.
procumbens have more
anti-inflammatory activity than C.
gigantea at individual and together with Ibuprofen. It is therefore, suggested
that the extract probably possesses anti-inflammatory activity and may
inhibit the release or synthesis of various inflammatory mediators.

Conclusion

������ The standard drug Ibuprofen presented
a dose-dependent anti-inflammatory activity at all the dose regimens selected
for the treatment after the carrageenin injection. The Ibuprofen also seems
to be sensitive to clycooxygenase inhibitors. The aqueous extract of T.
procumbens andmethanolic
extract of C. gigantea inhibited edema during all phases of
inflammation. The extracts also increased the inhibition of edema if treated
with the standard drug Ibuprofen. The outcome of the experiment is on the
expected, it probably inhibited the different aspects and chemical mediators
of inflammation and has been used to evaluate the effect of non-steroidal
anti-inflammatory agents.

Acknowledgment

����� The authors are thankful to the
National Botanical Research Institute, Lucknow, India, for taxonomical
identification of plant materials.